My long-term career plan is to become an independent physician-scientist investigator focused on the synthesis and utilization of nanotechnology systems for needle-free transcutaneous immunotherapeutics. My immediate career objective is to obtain the skill sets necessary to become scientifically independent. The research goal of this K08 Mentored Clinical Science Research Career Development proposal is to develop a novel, nontoxic, biodegradable, nanopolymersome (NPS)-based vaccine delivery system that will topically co- deliver antigen/adjuvant for transcutaneous immunization (TCI). Our novel approach has the potential to revolutionize vaccine strategies by addressing two scientific barriers: 1) increasing the versatility and efficacy of current vaccines by enhancing cell-mediated immunity through targeting of the epidermal Langerhans cells (LCs), and 2) enabling existing vaccine antigens to be co-delivered with adjuvant by a needle-free route. This NIH K08 grant will specifically contribute to my career development by providing the framework, mentorship, and protected research time enabling me to obtain the skills necessary to become scientifically independent. These skill sets include: obtaining a knowledge base in immunology and nanoparticle delivery systems; developing research skills in methodology, experimental design, and data analysis; developing leadership qualities for lab management; effective publication strategies; and maintenance of clinical dermatology expertise. I will work on this research program under the co-mentorship of Noah Craft, MD, PhD, and Samir Mitragotri, PhD, experts in immunotherapeutics and nanoparticle delivery systems for TCI, respectively. The institutional commitments from the Department of Medicine at Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, the Department of Engineering at UCSB, and the California NanoSystems Institute (CNSI) at UCLA, will support me to fulfill my ambition and career objectives. The World Health Organization estimates that 5-10% percent of the 16 billion injections administered worldwide each year are given for immunization. Thirty percent of those injections have been determined to be unsafe and account for 33% of HBV, 40% of HCV, and 5% of all new HIV infections. The direct cost associated with this significant morbidity and mortality has been estimated to be 1.4 billion US dollars annually. In hopes of obviating the risk of significant injuries associated with these injections, needle-free immunization has become a central initiative in global health care. I hypothesize that the integration of vaccine adjuvant and the encapsulation of antigen into the NPS can be achieved in a predictable and stepwise manner resulting in an effective transcutaneous vaccine. The antigen/adjuvant co-delivery NPS comprised entirely of an amphiphilic diblock copolymer poly(ethylene oxide) (PEO) and polycaprolactone (PCL), will penetrate the stratum corneum to reach the vital epidermis and induce an antigen specific immune response. After optimization, I will use this approach to generate L1 capsid/IMQ NPSs and hypothesize that this vaccine will induce a protective immune response against papilloma development in a Cottontail Rabbit Papilloma Virus (CRPV) rabbit model. To test these hypotheses, I propose the following aims: AIM 1: Construct a biodegradable NPS-based delivery system that integrates/encapsulates the model antigen ovalbumin (OVA) and the adjuvant Toll-Like-Receptor-7 (TLR7)-agonist imiquimod (IMQ), generating OVA/IMQ-NPSs. While previous research has focused on liposomal, peptide, and protein delivery systems, investigation on more advanced nanotechnology systems remains in a nascent stage. I will characterize and evaluate the feasibility of integrating IMQ into the membrane while encapsulating OVA into the aqueous core. AIM 2: Determine the optimal physico-chemical properties of NPSs for skin penetration and LC activation, and the mechanisms underlying OVA-specific immune induction by NPS. I will test NPSs varying in copolymer composition, particle size, deformability, and mechanical toughness to optimize this delivery system. I will monitor the ability of NPSs to traverse the stratum corneum, deliver, and release the payload to the epidermal LCs, and track loaded LC migration to the lymph node in vivo. I will also determine the underlying immune mechanisms generated by NPSs and evaluate the functional properties of the NPS vaccine co-delivery system that are required for induction of OVA-specific CD8+ T cell responses and humoral responses in vivo. AIM 3: Determine the ability of L1 capsid/IMQ-NPSs to generate a protective immune response against CRPV challenge and papilloma development in vivo. As a model of disease prevention in humans, I will test the ability of NPS to deliver the L1 capsid protein from CRPV with IMQ to generate a protective immune response against CRPV challenge and papilloma development. This rabbit model accurately reflects HPV infection in humans allowing for evaluation of humoral and cellular immune responses to TCI with loaded NPS. Summary: If this model is successful, it will provide a basis for collaborations with vaccine development teams to transform existing needle-based vaccine systems to needle-free TCI and translation to human use. During this K08 phase, I will have developed a tractable system that will serve as a valuable tool for TCI and other investigative applications. As a future independent investigator, this system will enable me to collaborate with scientists and clinicians to address a wide scope of challenges in medicine.